We have continued our investigations of the determinants of G-protein-coupled receptor (GPCR) selectivity for G-protein using recombinant expressed GPCRs and G-protein alpha subunit chimeras to map the contact site(s) on the alpha subunit responsible for receptor-selective interactions. Previously, we had constructed chimeras between G-alpha-i1 and G-alpha-q containing differing lengths of G-alpha-q sequence in place of the C-terminal sequence of G-alpha-i1 and testing for capacity to be activated by the human 5HT1A and 5HT2c receptors which are uniquely selective for the respective G-alpha-i1 and -q subunit types. Those experiments mapped the selective contact site(s) to include at least 50 residues of the carboxyl terminus of G-alpha-q. By co-expression of the chimeric G-alpha constructs with the yeast N-myristoyltransferase enzyme in E. coli hosts and purifying the myristoylated (myr-G-alpha) proteins to homogeneity, we have now refined this mapping. Our current constructs include chimeric G-alpha subunits containing G-alpha-q sequence internal to the C-terminus. When purified to homogeneous myr-G-alpha products these constructs show that sequence provided by the beta5 and beta6 strands of the protein contribute high affinity interaction with 5HT2c receptor while the full 50 amino acid residues of the carboxyl terminus is required for both high affinity and maximal efficacy of 5HT2c activation. [unreadable] We have also examined the role of the lipid modification of the G-alpha-i1/q chimeric subunit chains in receptor interaction by separating the non-myr- and myr-G-alpha products. In each case, only the myr-alpha was capable of receptor activation, either by 5HT1A receptors or cephalopod rhodopsin for the wild-type G-alpha-i1 or 5HT2c receptors and cephalopod rhodopsin for the G-alpha-i1/q chimeric constructs. Further, chimeric constructs of G-alpha-s containing G-alpha-i1 sequence replacing the N-terminal 37 residues expressed in E. coli allowed us to perform a similar experiment testing beta2-adrenergic receptor (b2AR) activation of unmodified and lipid-modified G-alpha-s. As for the G-alphai1/q chimeras, only the myr-G-alphai1/s chimeras were activated by the b2AR. These experiments also determined that the contact(s) between the G-alpha-s constructs and b2AR involved at least three distinct sites on the G-alpha chain, since rotations of the amino terminal G-alphai1 alpha helix (producing stereo-enantiomers) disrupted activation by the b2AR while preserving the capacity of the G-alpha chains to bind GTP and interact with the G-beta-gamma dimer. Further, chimeric constructs similar to the G-alphai1/q set with G-alpha-s sequence at the carboxyl terminus define the same selective contact surface for b2AR as found for G-alphai1/q and 5HT2c receptor (manuscript in preparation).